Cell adhesion molecules (CAMs) constitute a group of proteins mediating adhesion between cells. A major group of CAMs belongs to the immunoglobulin (Ig) superfamily characterised by the presence of immunoglobulin domains. The neural cell adhesion molecule (NCAM) is such a cell adhesion molecule of the Ig superfamily that is particularly abundant in the nervous system. NCAM is expressed on the external membrane of nerve cells. When an NCAM molecule on one cell binds to another NCAM molecule on another cell (homophilic binding), the binding between the two cells is strengthened. NCAM not only binds to NCAM but also to other proteins and/or glycoconjugates found on nerve cells or in the extracellular matrix (heterophilic binding). NCAM also binds ATP. NCAM interactions influence migration of cells, extension of neurites, fasciculation of neurites, cell proliferation, cell survival, and formation of synapses.
NCAM is encoded by a single gene, containing at least 25 exons. Due to alternative splicing of precursor mRNA, a variety of mature mRNA species and thereby protein isoforms of NCAM can be produced. Three major NCAM isoforms are generated by alternative splicing of exons 15 and 18 determining the mode of attachment of NCAM to the plasma membrane and the size of the intracellular NCAM domains, respectively. In the nervous system a glycosylphosphatidyl inositol (GPI) anchored 120 kDa isoform is expressed on the surface of glial cells, a transmembrane 140 kDa isoform is expressed on both neurons and glial cells, whereas a transmembrane 180 kDa isoform is found predominantly on the surface of neurons. The extracellular part of NCAM comprises five Ig-like homology modules (Ig1, Ig2, Ig3, Ig4 and Ig5) and two fibronectin type III modules (F3,1 and F3,2) (Berezin et al., 2000).
Heterophilic ligands of NCAM comprise a variety of heparan sulfate proteoglycans (e.g. agrin) and chondroitin sulfate proteoglycans (e.g. neurocan). NCAM Ig1 and Ig2 are probably the structural determinants of the interaction of NCAM with heparan sulfate proteoglycans since these two modules have been shown to bind heparin (Kiselyov et al. 1997). Reports on whether the core protein or the carbohydrate moieties are responsible for the binding of proteoglycans to NCAM are contradictory, and the contribution of this interaction to NCAM-mediated cellular functions is currently not understood (Retzler et al. 1996). The neural cell adhesion molecule L1 and the fibroblast growth factor (FGF) receptor are other heterophilic ligands of NCAM. The interaction between NCAM and L1 has been shown to be mediated by N-linked oligo-mannosidic glycans carried by L1 and a lectin-like binding site localised in the fourth Ig module of NCAM. Through this binding NCAM has been suggested to participate in a so-called assisted L1-L1 homophilic interaction (Horstkorte et al., 1993) presenting an interesting example of co-operation between two neural CAMs.
Three different models of homophilic binding have been suggested: 1) a binding between the third Ig-like modules (Rao et al., 1992) of two opposing molecules; 2) involvement of all five Ig-like modules in an antiparallel interaction (Ranheim et al., 1996); and 3) a reciprocal binding of the first and second Ig-like modules (Kiselyov et al., 1997). The latter model has recently been confirmed by nuclear magnetic resonance (NMR) analysis (Jensen et al., 1999) and X-ray crystallography (Kasper et al., 2000).
NCAM plays a crucial role during the development of the nervous system and of organs, such as kidney, bowel, heart, gonads, pancreas, and muscles. In the mature nervous system NCAM is important for the plasticity of neuronal connections associated with regeneration, learning and memory. In the peripheral nervous system NCAM is involved in the initiation of outgrowth of nerve fibres and formation of nerve-muscle connections in regeneration after damage including lesions.
In signal transduction NCAM transduces extracellular signals leading to tyrosine phosphorylation, such as for example of the FGF-receptor, and an increase in intracellular calcium concentration.
Doherty and Walsh (1999) describe that NCAM, N-cadherin and L1 stimulate axonal growth by activating the fibroblast growth factor receptor (FGFR) in neurons.
NCAM binding compounds capable of stimulating differentiation and/or neurite outgrowth from cells presenting NCAM are disclosed in WO 00/18801, in which the compounds are used in the treatment for regeneration of NCAM presenting cells.
The identification of one such compound, C3, is described by Rønn et al. (1999). C3 stimulates outgrowth by activating a signalling pathway identical to that activated by homophilic NCAM binding, but it does not bind directly to FGF receptors.
Various factors may cause neuronal cell death. Preventing neuronal cell death in individuals being exposed to risk factors causing cell death may be called maintaining/stimulating or promoting survival of the cells, or it may be called neuroprotection.
When neuronal cells are damaged, e.g. by reduced oxygen supply, the processes of cell death start and lead to cellular dysfunction, “collapse” of the intercellular communication between cells (network), retraction of cell processes and eventually cell death. Preventing neuronal cell death, i.e. stimulating/promoting survival means that the cells are protected from initiation of the processes of cell death.
Survival of nerve cells has been discussed in some references, for example Hulley et al. (1998) disclose that the L1 neural cell adhesion molecule is capable of stimulating survival and differentiation in fetal mid-brain dopaminergic neurons cultured in the presence of the toxin MPP+.
U.S. Pat. No. 6,037,320 describes the identification of a neurotrophic factor, NT-4 and in U.S. Pat. No. 5,767,240 an activity-dependent neurotrophic factor capable of increasing the survival of spinal cord neuronal cells, cerebral cortical cells and hippocampal neurons is revealed.
Further, U.S. Pat. No. 5,567,682 concerns a method of treating the symptoms of Alzheimer's disease by intranasal administration of short chain peptides. The peptides promote neuronal survival by reducing or halting progressive neuronal degeneration.
NCAM has recently been demonstrated to have an ecto-adenosine triphosphatase (ATPase) activity (Dzhandzhugazyan and Bock, 1993 and 1997). The role of this activity in ATP is one of the most abundant neurotransmitters in the nervous system. In a recent study it has been demonstrated that ATP modulates NCAM induced neurite outgrowth, indicating that ATP may be a regulator of the putative NCAM-FGF-receptor signalling pathway (Skladchikova et al., 1999).
However, the inventors of the present invention have surprisingly found that a compound comprising the third Immunoglobulin (Ig3) module, and/or the fourth immunoglobulin (Ig4) module, and/or the fifth Immunoglobulin (Ig5) module, and/or the first Fibronectin III (Fn3,1) module, and/or the second Fibronectin III (Fn3,2) module of neural cell adhesion molecule (NCAM), or a fragment, or a variant thereof is capable of inducing differentiation, modulating proliferation, stimulating regeneration, neuronal plasticity and survival of cells through an interaction with the Fibroblast Growth Factor (FGF) receptor and/or adenosine-tri-phosphate (ATP) and/or L1.